## [1] TRUE

[]{CaRpools.png)

Screen

Screen Information
Screen ID PILOT001
Screening Date 2013-11-11 to 2013-11-23
Organism Homo sapiens
Cell Line HCT116, HCT116 cells were stably transfected with CAS9-GFP (vector: LOTTA). This was done via lentiviral transduction (MOI not determined). Cells were then selected with puromycin. Cells were FACS sorted 2X for homogenous and high GFP, i.e. CAS9 expression.
Drug KillerTRAIL 50 ng / mL
Control DMSO 0,001%
Number of Target ca. 800
Designs per Gene varies: 10-20
Library 12K lib, see annotation file (fasta); sgRNA design was based on very simple design rules (G_N19; before NGG PAM) since parameters that can predict efficacy were not known when the oligo lbrary was designed and ordered.
Library Reference File pilot-screen-library
NGS MiSeq, 100 bp single end, Miseq V2 Kit

MIACCS
The MIACCS-file can be found at

./data/

MIACCS.xls

Description
Apoptosis related genes/Kinome Library in HCT116 Cas9-GFP cells. TRAIL resistance screen.

Plasmid Overview

Plasmid Overview

Plasmid Overview

Experimental Setup

Load Data

Stats

All file-based output (e.g. tables) is stored in:
./data.

General

General stats can be found in
TRAILscreen-STATS.xls.
The following read count statistics were calculated for the single datasets.

Readcount Control #1 Control #2 TRAIL #1 TRAIL #2
Mean 79 108 109 109
Median 51 74 41 39
SD 90 115 244 260
Min 0 0 0 0
Max 1141 1304 6819 8257
# sgRNA not present 934 817 1563 1573

Missing sgRNAs in Datasets

Information of how many sgRNA per gene were not present in the mapped datasets is stored in:

./data/

TRAILscreen_DROPOUT.xls

In brief, the following number of sgRNAs had a read count of 0 in the dataset:

Control #1: 934 missing sgRNAs

Control #2: 817 missing sgRNAs

TRAIL #1: 1563 missing sgRNAs

TRAIL #2: 1573 missing sgRNAs

All Stats

In depth dataset read count stats can be found in

./data/

TRAILscreen-STATS.xls

Quality Control

Read Distribution

These plots show how the read count of the sgRNAs for each dataset is distributed. Depending on the treatment stringency, e.g. in resistance or dropout screens, the data can show asymmetry. However, the major population should be more or less normally distributed.

Read Depth

The following plot shows the read count for each gene normalized to the number of sgRNAs. Spikes indicate a higher read count per sgRNA for this particular gene.
One would expect no outstanding spikes within the untreated data samples, however spikes within the treated datasets indicate a read count enrichment for this particular gene.
If a non-targeting control has been set in the MIACCS file, this control is highlighted in orange color.

Designs per Gene

These plots provide an overview of the representation of sgRNAs per gene within your data.
Depending on the number of sgRNAs per gene in the library, one would expect a representation of more than 80 % of sgRNAs per gene in the untreated samples. Moreover, genes with a low percentage of present sgRNAs will also show a reduced readcount.

Controls

Non-Targeting

Non-targeting controls are sgRNAs that do either not target the genome at all (so called random or scramble sgRNAs) or target a gene that does not show a phenotype in the screen.
Therefore, the scatter for these, which are highlighted in blue, will be distributed within the main cloud of scatter points.

Non-targeting control: random

Positive Controls

SgRNAs targeting genes that will show a phenotype in the screening setup can be used as positive controls.
These will show either an enrichment (in resistance screens) or a depletion (in dropout screens) in the treatment.
Within the scatter

Positive Control: CASP8

Hit Analysis

Hit analsysis is performed using three different methods:

For each analysis method, separate plots will be created and analysis files will be written to TRAILscreen_HIT-CALLING.xls. See below for further information.

The following adjusted p-values are used to determine significance levels:

Method p-value
Wilcox 0.05
DESeq2 0.001
MAGeCK 0.05

Wilcox

Within this approach, the read counts of all sgRNAs in one dataset are first normalized by the function set in the MIACCS file. By default, normalization is done by read count division with the dataset median.
Then, the fold change of each population of sgRNAs for a gene is tested against the population of either the non-targeting controls or randomly picked sgRNAs, as defined by the random picks option within the MIACCS file, using a two-sided Mann-Whitney test with FDR correction.

DESeq2

For the DESeq2 analysis implementation, the read counts of all sgRNAs for a given gene are first summed up to increase the available read count.
Then, DESeq2 analysis is perfomed, which includes the estimation of size-factors, the variance stabilization using a parametric fit and a Wald-Test for differnece in log2 fold changes between the untreated and treated data.
More information about this can be found in Love et al.
Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2
Genome Biology 2014

MAGeCK

MAGeCK analysis uses a rank-based model to test for a change in abundance of sgRNAs after median normalization of the dataset.
Further information can be found at the MAGeCK Homepage.

Wilcox

All analysis data can be found in the TRAILscreen_HIT-CALLING.xls file.

P-Value Distribution

For all genes present in the data, the -log10 corrected p-values are plotted to estimate how well the analysis method performed for this screen. A straight line of points with only small differences in the p-value indicates that the analysis method did not perform well. Genes that resulted in a p-value below the threshold set in the MIACCS file are highlighted in red color.

Enriched

The following genes showed enrichment in the treatment datasets with a p-value smaller than 0.05:

untreated treated foldchange p.value
BAX 3.747567 21.346353 5.542859 0.0178241
CASP8 4.037869 41.292318 7.151636 0.0178241
PASK 2.632026 7.833771 2.575938 0.0178241
FADD 3.765291 35.004169 7.126894 0.0184341

According to the value set, the top 5 % enriched genes were:

untreated treated foldchange p.value
BAX 3.747567 21.346353 5.542859 0.0178241
CASP8 4.037869 41.292318 7.151636 0.0178241
PASK 2.632026 7.833771 2.575938 0.0178241
FADD 3.765291 35.004169 7.126894 0.0184341
FN3K 2.394351 6.545106 2.767443 0.4030853
SPA17 2.616958 5.136043 1.866767 0.4030853
CDK5RAP3 2.414025 4.678275 1.865306 0.4202992
TNFRSF11A 2.664088 6.650262 1.597737 0.7848917
IP6K3 2.313419 5.295556 2.520348 0.8019777
ABI1 2.999779 4.458453 1.463144 0.9671744
ADCK1 3.013987 5.668811 1.713397 0.9671744
AKAP6 2.124669 3.065627 1.367359 0.9671744
ALPK2 2.424144 4.293484 1.749312 0.9671744
AMHR2 2.206300 7.288762 2.761481 0.9671744
AURKA 2.668363 3.391432 1.368951 0.9671744
AVPR1B 2.980878 4.610329 1.469106 0.9671744
AXL 3.189164 6.108251 1.481180 0.9671744
BCL2L1 2.403153 4.073267 1.504323 0.9671744
BCL2L11 2.486840 2.879482 1.310120 0.9671744
BCL2L14 1.798592 2.359792 1.252899 0.9671744
BCL6B 2.150004 3.387478 1.557439 0.9671744
BID 1.984433 3.494241 1.451740 0.9671744
BIRC3 1.266241 1.677194 1.297998 0.9671744
BLK 2.414149 4.606897 1.694439 0.9671744
BMP2K 2.725432 7.364192 2.114134 0.9671744
BMPR1A 2.851042 11.049698 3.242482 0.9671744
BMPR1B 2.574995 6.898140 1.993310 0.9671744
BNIP2 1.795370 3.609330 1.824645 0.9671744
BNIP3 2.280309 2.488743 1.032476 0.9671744
CALM3 2.950194 8.018547 2.108928 0.9671744
CAMK1G 3.038230 3.284475 1.063666 0.9671744
CAMK4 3.120206 4.176575 1.298515 0.9671744
CAMKK2 3.096820 5.179529 1.571313 0.9671744
CARD14 2.505785 4.410819 1.693491 0.9671744
CASK 2.442766 3.036390 1.270888 0.9671744
CDC42BPA 1.765662 2.496069 1.478136 0.9671744
CDK1 2.480657 4.789994 1.816161 0.9671744
CDK10 2.357760 2.987199 1.135816 0.9671744
CDK17 2.000075 7.985030 2.652919 0.9671744
CDK8 2.849182 4.568695 1.020590 0.9671744
CINP 1.700964 2.942366 1.646489 0.9671744

Depleted

The following genes showed depletion in the treatment datasets with a p-value smaller than 0.05:

No genes showed significant depletion with a p-value lower than 0.05

According to the value set, the top 5 % depleted genes were:

untreated treated foldchange p.value
MVD 2.437008 2.045153 0.8590363 0.4030853
CDC42BPG 2.457555 2.076689 0.8770324 0.8749233
AK1 2.354821 2.215271 0.9615239 0.9671744
ALPK3 1.759417 1.664550 0.9308875 0.9671744
AZU1 2.965272 2.961382 0.9237615 0.9671744
BBC3 2.429212 2.246981 0.9394726 0.9671744
BCL2L12 3.872529 3.402968 0.9480240 0.9671744
BCL3 2.893494 2.714992 0.9531555 0.9671744
BCL9 1.593733 1.361372 0.8862743 0.9671744
CAMK2G 2.710180 2.687168 0.9540536 0.9671744
CDKN1B 2.097322 1.966672 0.9655601 0.9671744
CHEK1 2.554584 2.300618 0.9370998 0.9671744
CSF1R 3.466243 3.247571 0.9083817 0.9671744
DFFA 2.417806 2.465291 0.9844160 0.9671744
DGCR14 3.287838 3.033271 0.9585501 0.9671744
DUSP2 2.424193 2.163852 0.9824662 0.9671744
DUSP6 2.394930 2.475443 0.9775693 0.9671744
FASTK 2.429278 2.201806 0.9555991 0.9671744
GMFB 2.359733 2.326454 0.9807980 0.9671744
GSK3A 1.900868 1.941428 0.9971515 0.9671744
ITPKA 2.607611 2.165611 0.9336513 0.9671744
MAPK3 2.309495 2.065603 0.9843943 0.9671744
MAPK7 2.961058 2.784324 0.9896817 0.9671744
MPP3 1.740669 1.618087 0.9367596 0.9671744
NEK8 2.423821 2.357821 0.9888161 0.9671744
NRG3 2.387979 2.226621 0.9159026 0.9671744
NTRK1 2.441251 2.275309 0.9450689 0.9671744
PAPSS2 2.400848 2.199667 0.9570816 0.9671744
PDK4 1.965118 1.889239 0.9622144 0.9671744
PFKFB2 1.875278 1.848614 0.9928155 0.9671744
PIK3R3 2.401635 2.197287 0.9221953 0.9671744
PKMYT1 3.075614 3.038899 0.9667207 0.9671744
PRKACB 2.211535 1.700751 0.8653760 0.9671744
PRKCE 2.877672 2.620680 0.9241182 0.9671744
PRKCQ 1.925311 1.762508 0.9351715 0.9671744
PTK6 3.124685 3.441370 0.9576040 0.9671744
RIPK1 3.451535 3.266084 0.9424100 0.9671744
RPS6KC1 2.023408 2.026696 0.9733447 0.9671744
SCYL3 2.114794 1.976152 0.9914523 0.9671744
SGK2 3.128786 2.851614 0.8730285 0.9671744
SHC1 4.391180 3.819887 0.9400818 0.9671744

DESeq2

All analysis data can be found in the TRAILscreen_HIT-CALLING.xls file.

P-Value Distribution

For all genes present in the data, the -log10 corrected p-values are plotted to estimate how well the analysis method performed for this screen. A straight line of points with only small differences in the p-value indicates that the analysis method did not perform well. Genes that resulted in a p-value below the threshold set in the MIACCS file are highlighted in red color.

Enriched

The following genes showed enrichment in the treatment datasets with a p-value smaller than 0.001:

log2FoldChange lfcSE padj sgRNA
CASP8 2.8539899 0.1968984 0.0000000 2
FADD 2.7466780 0.2008926 0.0000000 4
BAX 2.0761255 0.1951457 0.0000000 2
RPS6KA5 2.0613922 0.1993450 0.0000000 5
CDK17 1.9749507 0.2098786 0.0000000 6
HRK 1.7832227 0.1986851 0.0000000 0
TP53 1.7263896 0.1990543 0.0000000 3
BMPR1A 1.6606699 0.1934715 0.0000000 2
AMHR2 1.5964273 0.2040561 0.0000000 3
PRKAR1A 1.3066797 0.1966018 0.0000000 2
PASK 1.3056495 0.2018553 0.0000001 2
PRKCH 1.2768183 0.2034549 0.0000003 4
PRKD3 1.2469315 0.2049320 0.0000009 4
PTPRR 1.1714789 0.1988912 0.0000031 2
FN3K 1.2257973 0.2101826 0.0000044 2
BMP2K 1.1356520 0.1969679 0.0000065 2
MDM2 1.1155550 0.2005697 0.0000212 3
PRKCZ 1.1267946 0.2027299 0.0000217 2
PAG1 1.2017977 0.2167288 0.0000233 3
BMPR1B 1.1476671 0.2081757 0.0000279 0
PRKY 1.1629272 0.2119197 0.0000322 1
MYC 1.0493447 0.1952001 0.0000603 4
DUSP4 1.0685262 0.2020427 0.0000972 5
CSNK2A2 1.3577535 0.2578919 0.0001106 2
LMTK3 1.0154113 0.1953558 0.0001587 3
MAP2K7 1.1032489 0.2128120 0.0001706 1
LATS1 1.2426869 0.2460336 0.0003452 1
CALM3 1.0844637 0.2148274 0.0003499 2
IP6K3 0.9775242 0.1947187 0.0004042 2
RIPK2 0.9771641 0.1981684 0.0006398 2

According to the value set, the top 5 % enriched genes were:

log2FoldChange lfcSE padj sgRNA
CASP8 2.8539899 0.1968984 0.0000000 2
FADD 2.7466780 0.2008926 0.0000000 4
BAX 2.0761255 0.1951457 0.0000000 2
RPS6KA5 2.0613922 0.1993450 0.0000000 5
CDK17 1.9749507 0.2098786 0.0000000 6
HRK 1.7832227 0.1986851 0.0000000 0
TP53 1.7263896 0.1990543 0.0000000 3
BMPR1A 1.6606699 0.1934715 0.0000000 2
AMHR2 1.5964273 0.2040561 0.0000000 3
PRKAR1A 1.3066797 0.1966018 0.0000000 2
PASK 1.3056495 0.2018553 0.0000001 2
PRKCH 1.2768183 0.2034549 0.0000003 4
PRKD3 1.2469315 0.2049320 0.0000009 4
PTPRR 1.1714789 0.1988912 0.0000031 2
FN3K 1.2257973 0.2101826 0.0000044 2
BMP2K 1.1356520 0.1969679 0.0000065 2
MDM2 1.1155550 0.2005697 0.0000212 3
PRKCZ 1.1267946 0.2027299 0.0000217 2
PAG1 1.2017977 0.2167288 0.0000233 3
BMPR1B 1.1476671 0.2081757 0.0000279 0
PRKY 1.1629272 0.2119197 0.0000322 1
MYC 1.0493447 0.1952001 0.0000603 4
DUSP4 1.0685262 0.2020427 0.0000972 5
CSNK2A2 1.3577535 0.2578919 0.0001106 2
LMTK3 1.0154113 0.1953558 0.0001587 3
MAP2K7 1.1032489 0.2128120 0.0001706 1
LATS1 1.2426869 0.2460336 0.0003452 1
CALM3 1.0844637 0.2148274 0.0003499 2
IP6K3 0.9775242 0.1947187 0.0004042 2
RIPK2 0.9771641 0.1981684 0.0006398 2
PRPS1 1.0154924 0.2099970 0.0010360 2
MAPK8IP2 0.9554903 0.2044514 0.0023104 2
CSNK2A1 0.9415114 0.2038046 0.0029940 5
PRKAG3 0.9230088 0.2009259 0.0033866 5
NEK11 0.9335366 0.2033509 0.0034314 3
TGFBR1 0.9607698 0.2096900 0.0035763 2
TNFRSF11B 0.9509991 0.2077597 0.0036486 0
MAPK13 0.9602742 0.2100303 0.0037378 3
MBIP 0.9025821 0.2012146 0.0056186 5
SEPHS1 0.8714230 0.1947709 0.0059241 1
TNFRSF11A 1.0021224 0.2284252 0.0088570 1

Depleted

The following genes showed depletion in the treatment datasets with a p-value smaller than 0.001:

log2FoldChange lfcSE padj sgRNA
SMG1 -1.429982 0.2123290 0.0e+00 7
PRKACB -1.326844 0.2187738 1.1e-06 4

According to the value set, the top 5 % depleted genes were:

log2FoldChange lfcSE padj sgRNA
SMG1 -1.4299818 0.2123290 0.0000000 7
PRKACB -1.3268439 0.2187738 0.0000011 4
DUSP2 -0.8839585 0.2036127 0.0109028 2
MAPK3 -0.8874385 0.2055074 0.0120925 4
MVD -1.0079883 0.2337749 0.0124369 2
HK3 -0.8896877 0.2091965 0.0161867 2
BCL2L12 -0.8493349 0.2009970 0.0182295 0
ROCK1 -0.8921263 0.2144381 0.0241872 3
WIF1 -0.8396864 0.2018872 0.0242731 3
BBC3 -0.7995261 0.2002468 0.0491891 2
SGK2 -0.7987094 0.2001047 0.0493829 2
PRKCQ -0.8650269 0.2190015 0.0587241 4
CDC42BPG -0.9735155 0.2476437 0.0633304 1
NRG3 -0.7790845 0.1998423 0.0723033 3
MPP3 -0.8573709 0.2218129 0.0825529 4
CDKN1B -0.7810259 0.2039736 0.0954487 2
MAPK7 -0.7443061 0.1956164 0.1051081 1
AK1 -0.7584758 0.1999155 0.1097052 2
CSF1R -0.7419327 0.1957882 0.1115685 1
ITPKA -0.9804692 0.2588762 0.1123445 1
CHEK1 -0.8384986 0.2227553 0.1231337 5
FASTK -0.8297426 0.2226460 0.1427661 1
SHC1 -0.8360697 0.2249526 0.1483839 0
ALPK3 -0.7916102 0.2130996 0.1493037 2
SCYL3 -0.7831114 0.2119499 0.1613076 3
C9orf96 -0.7498465 0.2060267 0.1990927 0
PIK3R3 -0.8057038 0.2266208 0.2741052 3
DGKG -0.7204221 0.2027228 0.2753605 5
PRKCE -0.7937112 0.2241867 0.2892502 2
PRKG2 -0.8289301 0.2356366 0.3145684 7
MASTL -0.7723763 0.2220390 0.3629619 4
BCL3 -0.7351915 0.2172953 0.5119468 1
LCK -0.6660242 0.1988919 0.5781373 1
NTRK1 -0.7545015 0.2277952 0.6563060 0
NEK8 -0.6734080 0.2042739 0.6928860 3
TLR1 -0.7084222 0.2149545 0.6941480 2
PDK4 -0.7204333 0.2189604 0.7067081 5
TNFRSF4 -0.8135550 0.2473382 0.7082275 1
PIK3CB -0.7376748 0.2258552 0.7653496 3
DGCR14 -0.7534509 0.2320389 0.8173098 5
PKMYT1 -0.6395897 0.1974689 0.8398297 0

MAGeCK

All analysis data for MAGeCK can be found in the TRAILscreen_HIT-CALLING.xls file.

P-Value Distribution

For all genes present in the data, the -log10 corrected p-values are plotted to estimate how well the analysis method performed for this screen. A straight line of points with only small differences in the p-value indicates that the analysis method did not perform well. Genes that resulted in a p-value below the threshold set in the MIACCS file are highlighted in red color.

Enriched

The following genes showed enrichment in the treatment datasets with a p-value smaller than 0.05:

pos rank.pos neg rank.neg sgrna.neg.good sgrna.pos.good
CASP8 0.001650 1 0.994410 804 0 10
BAX 0.001650 2 0.994232 775 1 9
FADD 0.001650 3 0.994410 792 1 9
PASK 0.003713 4 0.994410 800 1 11

According to the value set, the top 5 % enriched genes were:

pos rank.pos neg rank.neg sgrna.neg.good sgrna.pos.good
CASP8 0.001650 1 0.994410 804 0 10
BAX 0.001650 2 0.994232 775 1 9
FADD 0.001650 3 0.994410 792 1 9
PASK 0.003713 4 0.994410 800 1 11
TYRO3 0.659241 5 0.981079 109 4 9
AMHR2 0.659241 6 0.994410 796 1 5
FN3K 0.661245 7 0.994232 730 2 11
PRKY 0.785149 8 0.994232 479 4 8
CDK5RAP3 0.785149 9 0.994450 807 1 9
ROR2 0.785149 10 0.994232 734 3 7
BMPR1A 0.785149 11 0.994232 637 3 6
DGKI 0.785149 12 0.994232 609 4 5
IP6K3 0.785149 13 0.994232 678 2 10
SGK1 0.785149 14 0.994232 562 2 6
CSNK1A1 0.785149 15 0.993066 290 6 2
RPS6KA5 0.792340 16 0.994410 802 1 5
FGFR2 0.792340 17 0.994232 435 4 6
FER 0.792340 18 0.994232 707 2 8
GAK 0.792340 19 0.994232 527 2 7
MYC 0.819189 20 0.994232 740 2 8
NME7 0.819189 21 0.994232 359 3 9
MAP3K13 0.903465 22 0.993066 214 6 8
CDK17 0.903465 23 0.994232 533 1 6
UCK1 0.903465 24 0.994410 790 2 8
SPA17 0.903465 25 0.994232 781 2 9
STK32A 0.903465 26 0.994232 596 1 8
MVK 0.912769 27 0.994232 695 2 6
BIRC5 0.912769 28 0.993066 177 5 4
MAP2K7 0.912769 29 0.950888 62 7 7
TNFRSF8 0.919967 30 0.993066 225 4 6
RIPK2 0.987129 31 0.994232 524 3 7
MAP2K3 0.987129 32 0.993066 137 5 7
CDK9 0.987129 33 0.994232 493 3 5
DUSP10 0.987129 34 0.994232 633 1 8
LMTK3 0.987129 35 0.994232 673 2 4
TK2 0.987129 36 0.994232 602 3 9
NLK 0.987129 37 0.994232 663 2 8
PRKD2 0.987129 38 0.994232 444 5 7
PRPS1 0.987129 39 0.993066 247 4 6
SGK223 0.987129 40 0.993066 289 6 5
SRPK2 0.987129 41 0.994232 658 2 5

Depleted

The following genes showed depletion in the treatment datasets with a p-value smaller than 0.05:

No genes showed significant depletion with a p-value lower than 0.05

According to the value set, the top 5 % depleted genes were:

pos rank.pos neg rank.neg sgrna.neg.good sgrna.pos.good
TNFRSF13C 0.996031 555 0.701894 1 4 1
DYRK2 0.987129 373 0.701894 2 9 4
CDC42BPG 0.996304 803 0.701894 3 9 2
CHEK1 0.996304 777 0.701894 4 6 2
RAPGEF3 0.987129 78 0.701894 5 5 6
TNFRSF11A 0.987129 96 0.701894 6 9 1
CSF1R 0.996304 784 0.701894 7 8 1
PTK6 0.996304 743 0.701894 8 9 3
BMF 0.996304 585 0.701894 9 2 4
TESK2 0.987129 478 0.701894 10 6 4
NUAK2 0.987129 213 0.701894 11 3 5
AURKA 0.987129 219 0.701894 12 7 3
PRKACB 0.996304 769 0.701894 13 7 1
NRG3 0.996304 724 0.701894 14 8 3
TNFRSF13B 0.987129 344 0.701894 15 4 2
AZU1 0.996304 718 0.701894 16 6 3
WIF1 0.996304 799 0.701894 17 7 2
MAPK7 0.996304 678 0.701894 18 8 2
HK3 0.996304 650 0.701894 19 6 4
BCL2L12 0.996304 707 0.701894 20 6 2
TP53BP1 0.987129 395 0.701894 21 2 4
ITPKA 0.996304 795 0.701894 22 8 2
JAK3 0.987129 516 0.701894 23 8 3
CSNK1A1L 0.987129 451 0.736436 24 7 3
PAK7 0.987129 452 0.736436 25 7 5
MAP3K6 0.987129 350 0.790240 26 5 3
CAMK1G 0.996304 600 0.790240 27 6 3
PFKFB1 0.987129 340 0.790240 28 10 5
SGK2 0.996304 786 0.790240 29 6 1
PRKAA1 0.996304 697 0.790240 30 7 3
PKMYT1 0.996304 708 0.790240 31 9 3
TSSK3 0.996304 570 0.790240 32 6 3
NTRK3 0.987129 377 0.790240 33 7 3
FASTK 0.996304 703 0.790240 34 8 4
AK1 0.996304 757 0.790240 35 9 2
CLK3 0.996304 635 0.808760 36 9 3
MAPKAPK5 0.987129 514 0.808760 37 7 4
PTPRG 0.996304 624 0.808760 38 5 4
CDC42SE2 0.987129 517 0.808760 39 8 3
ACVRL1 0.996304 651 0.808760 40 6 4
PAPSS1 0.987129 527 0.808760 41 8 5

Hit Candidate Overview

Overview

Genes which showed enrichment or depletion within the individual analysis methods are presented in the following section.
All genes that showed significant enrichment within Wilcox, DESeq2 and MAGeCK with the given p-value cutoffs are highlighted in red color.
All genes that showed significant depletion within Wilcox, DESeq2 and MAGeCK with the given p-value cutoffs are highlighted in blue color.

Moreover, all genes that showed up as significantly enriched or depleted in any of the analysis methods are highlighted in orange color. Genes that showed no significant effect are presented in grey or black color.

Overlaps in Enrichment Analysis

The following genes showed enrichment in all three analysis methods.

Within the top enriched hits, the overlap of enriched hits per analysis method is displayed as follows:

Overlaps in Depletion Analysis

ATTENTION
No overlapping depleted genes were found between all three methods.

Therefore, the overlap from the 5 % of genes within all methods is used for plotting possible hit candidates.

We strongly advise you to have a closer look at the individual outputs of the analysis methods and to carefully look at the following candidate genes.

Within the top depleted hits, the overlap of depleted hits per analysis method is displayed as follows:

Hit Candidates

Scatterplots representing the gene read count as well as sgRNA read count for all overlapping hits are plotted in this section.

If there were signficantly enriched or depleted genes that overlapped in all analysis methods, they will be presented here.
Therefore, the top overlapping hits of each Hit Analysis with a p-value below the thresholds
for each analysis method are highlighted in orange within the scatter plots for all four samples.

In the case that no significantly enriched or depleted genes did overlap in all methods, those that overlapped within the 5 % of top enriched and the top 5 % depleted genes are used.
Therefore, the top overlapping hits of each Hit Analysis are highlighted in orange within the scatter plots for all four samples.

This allows a fast and easy view for single genes and its individual sgRNAs.
Moreover, individual sgRNA effects are plotted as well as the corresponding target sequence.

In this section, the following plots are generated for each hit candidate:

The scatter plots show the median normalized, log read count of each genes/sgRNA. Moreover, the blue lines indicate a read count foldchange of 2, the green lines indicate a read count foldchange of 4.

Enriched

CASP8

In addition to the plots below, the following information has been retrieved via biomaRt:

ENSEMBL ID (links to Ensembl) LRG_34

HGNC SYMBOL (links to GeneCards) CASP8

GENE DESCRIPTION
caspase 8, apoptosis-related cysteine peptidase [Source:HGNC Symbol;Acc:HGNC:1509]

GO TERM

MIM GENE DESCRIPTION
CASPASE 8, APOPTOSIS-RELATED CYSTEINE PROTEASE; CASP8;;MORT1-ASSOCIATED CED3 HOMOLOG; MACH;;FADD-HOMOLOGOUS ICE/CED3-LIKE PROTEASE;;FADD-LIKE ICE; FLICE;;MCH5

ENSEMBL PROTEIN ID
LRG_34p2

PROTEIN FAMILY DESCRIPTION
CASPASE PRECURSOR CASP APOPTOTIC PROTEASE MCH ICE APOPTOTIC PROTEASE [CONTAINS CASPASE SUBUNIT CASPASE SUBUNIT

Wilcox p-value: 0.01782408

DESeq2 p-value: 1.063499e-44

MAGeCK p-value: 0.00165

designs genes sequence log2Foldchange
2 CASP8_192_46512 CASP8 gatgatgcccttgtctccat 4.0442283
3 CASP8_193_46512 CASP8 gagatgtcagctcatagatg 4.0404115
9 CASP8_95_46512 CASP8 ggagacaagggcatcatcta 3.7513973
7 CASP8_199_46512 CASP8 gacatcgctctctcaggctc 3.5778575
10 CASP8_96_46512 CASP8 ggcatcatctatggcactga 3.4739444
6 CASP8_198_46512 CASP8 gcaaagtgactggatgtacc 2.1055251
1 CASP8_104_46512 CASP8 gcctgagagagcgatgtcct 1.7201422
5 CASP8_197_46512 CASP8 ggaaacacagttattcacag 1.6918053
11 CASP8_97_46512 CASP8 gagctgacatctcagttcac 0.8950126
4 CASP8_196_46512 CASP8 gcagaaagtcagcctcatcc 0.8529520
8 CASP8_218_46512 CASP8 gaaggcataaagcaagtttg 0.0000000
12 CASP8_99_46512 CASP8 ggagcaaccctatttagaaa -0.2440090

BAX

In addition to the plots below, the following information has been retrieved via biomaRt:

ENSEMBL ID (links to Ensembl) ENSG00000087088

HGNC SYMBOL (links to GeneCards) BAX

GENE DESCRIPTION
BCL2-associated X protein [Source:HGNC Symbol;Acc:HGNC:959]

GO TERM
mitochondrial outer membrane

MIM GENE DESCRIPTION
BCL2-ASSOCIATED X PROTEIN; BAX

ENSEMBL PROTEIN ID
ENSP00000375744

PROTEIN FAMILY DESCRIPTION
APOPTOSIS REGULATOR BAX

Wilcox p-value: 0.01782408

DESeq2 p-value: 1.593957e-23

MAGeCK p-value: 0.00165

designs genes sequence log2Foldchange
4 BAX_36_5982 BAX gtccaatgtccagcccatga 3.6370541
7 BAX_45_5982 BAX gcgtcccaaagtaggagagg 3.4780185
5 BAX_3_5982 BAX gaactgatcagaaccatcat 3.1659229
11 BAX_9_5982 BAX gcagaccgtgaccatctttg 2.6287417
8 BAX_46_5982 BAX gatggtcacggtctgccacg 2.4434385
1 BAX_10_5982 BAX gaccgtgaccatctttgtgg 2.4213382
6 BAX_44_5982 BAX gaggaggccgtcctggagac 1.9233456
3 BAX_18_5982 BAX gggggtaataaacctccttc 1.5871055
9 BAX_47_5982 BAX gagcactcccgccacaaaga 1.5148023
2 BAX_11_5982 BAX gctgaggcccccagctgcct -0.0430781
10 BAX_8_5982 BAX gacggcctcctctcctactt -0.3111825

FADD

In addition to the plots below, the following information has been retrieved via biomaRt:

ENSEMBL ID (links to Ensembl) LRG_228

HGNC SYMBOL (links to GeneCards) FADD

GENE DESCRIPTION
Fas (TNFRSF6)-associated via death domain [Source:HGNC Symbol;Acc:HGNC:3573]

GO TERM

MIM GENE DESCRIPTION
FAS-ASSOCIATED VIA DEATH DOMAIN; FADD;;FAS-ASSOCIATING PROTEIN WITH DEATH DOMAIN;;MORT1

ENSEMBL PROTEIN ID
LRG_228p1

PROTEIN FAMILY DESCRIPTION
FAS ASSOCIATED DEATH DOMAIN FAS ASSOCIATING DEATH DOMAIN CONTAINING MEDIATOR OF RECEPTOR INDUCED TOXICITY FADD

Wilcox p-value: 0.01843405

DESeq2 p-value: 1.205659e-39

MAGeCK p-value: 0.00165

designs genes sequence log2Foldchange
6 FADD_19_0 FADD gcagcatggagaagaggtct 4.3250217
1 FADD_10_3015 FADD gcaacctgacagagcgtgtg 4.0186830
5 FADD_18_0 FADD gaggcataggaacttgagct 3.6167997
7 FADD_20_0 FADD gctccagcagcatggagaag 3.2846277
8 FADD_21_0 FADD gtcgttctgctccagcagca 3.2504992
11 FADD_6_0 FADD gccgcttgcagaccccgcca 2.2663642
9 FADD_23_3015 FADD gcacacgctctgtcaggttg 2.0061298
12 FADD_9_0 FADD gctgctggagcagaacgacc 1.9549099
2 FADD_14_0 FADD gaaacggctgcggcttcgct 1.0323283
3 FADD_15_0 FADD gccgcttgcccagcggccca 0.3338738
10 FADD_4_0 FADD ggtggaatccttgggccgct -0.0800059
4 FADD_16_3015 FADD gtcctcgatgctgtcgatct -0.3712259

PASK

In addition to the plots below, the following information has been retrieved via biomaRt:

ENSEMBL ID (links to Ensembl) ENSG00000115687

HGNC SYMBOL (links to GeneCards) PASK

GENE DESCRIPTION
PAS domain containing serine/threonine kinase [Source:HGNC Symbol;Acc:HGNC:17270]

GO TERM

MIM GENE DESCRIPTION
PAS DOMAIN-CONTAINING SERINE/THREONINE KINASE; PASK;;PAS KINASE;;PASKIN;;KIAA0135

ENSEMBL PROTEIN ID
ENSP00000486149

PROTEIN FAMILY DESCRIPTION
UNKNOWN

Wilcox p-value: 0.01782408

DESeq2 p-value: 7.9517e-08

MAGeCK p-value: 0.003713

designs genes sequence log2Foldchange
14 PASK_110_25236 PASK gaatgagcagattcgtttta 2.5192130
12 PASK_10_6309 PASK ggccacgttagccggcaagg 2.3438587
2 PASK_0_18927 PASK gagcaccacacttcttccta 2.3183905
10 PASK_10_25236 PASK gcaagcacgacattaatcct 1.4918070
15 PASK_110_31545 PASK gccagcacatcccaaaggta 1.4773762
5 PASK_102_37854 PASK gatcagatcttttatgccct 1.4529940
4 PASK_100_31545 PASK gttgctaacgacaaagcttg 1.3469513
9 PASK_10_18927 PASK gccttggccaaggtcaacac 1.1772435
8 PASK_109_6309 PASK gaaataggcatggtgtgcgt 1.0279086
11 PASK_10_31545 PASK gcgtgagcttctggccaatc 1.0060151
13 PASK_110_0 PASK gctgacctgtcacctgtgag 0.5407919
6 PASK_106_0 PASK gtatatgctcatctgagtga 0.2050875
1 PASK_0_0 PASK ggatgctgagaagcctcacc 0.1610815
3 PASK_0_6309 PASK gatgcttctgtgccctggac 0.0893763
7 PASK_108_25236 PASK gaaagccaagacatcttcac -0.2255927

Depleted

CDC42BPG

In addition to the plots below, the following information has been retrieved via biomaRt:

ENSEMBL ID (links to Ensembl) ENSG00000171219

HGNC SYMBOL (links to GeneCards) CDC42BPG

GENE DESCRIPTION
CDC42 binding protein kinase gamma (DMPK-like) [Source:HGNC Symbol;Acc:HGNC:29829]

GO TERM
cell leading edge

MIM GENE DESCRIPTION
CDC42-BINDING PROTEIN KINASE, GAMMA; CDC42BPG;;MYOTONIC DYSTROPHY KINASE-RELATED CDC42-BINDING KINASE, GAMMA; MRCKG;;MRCK-GAMMA

ENSEMBL PROTEIN ID
ENSP00000345133

PROTEIN FAMILY DESCRIPTION
SERINE/THREONINE KINASE MRCK EC_2.7.11.1 CDC42 BINDING KINASE DMPK MYOTONIC DYSTROPHY KINASE RELATED CDC42 BINDING KINASE MRCK MYOTONIC DYSTROPHY KINASE

Wilcox p-value: 0.8749233

DESeq2 p-value: 0.06333045

MAGeCK p-value: 0.701894

designs genes sequence log2Foldchange
16 CDC42BPG_102_15130 CDC42BPG ggaccgtctcccgcccgagc 0.6054159
7 CDC42BPG_100_15130 CDC42BPG ggacacctccaactttgatg 0.4953713
5 CDC42BPG_100_0 CDC42BPG ggggaggggtgcagtgggcg 0.0525458
13 CDC42BPG_101_18156 CDC42BPG ggtcaccgtggtgaggcaga 0.0405820
6 CDC42BPG_100_12104 CDC42BPG ggagctggagcagctacgga 0.0139267
2 CDC42BPG_0_15130 CDC42BPG gttgtctggcttgacatccc -0.0573332
15 CDC42BPG_102_0 CDC42BPG gagggagtctggagaacaaa -0.2284839
4 CDC42BPG_0_9078 CDC42BPG gtagaggctgtgttggcagt -0.2611705
8 CDC42BPG_100_18156 CDC42BPG gtggtgaggcagagggacac -0.3513528
10 CDC42BPG_101_0 CDC42BPG gaacaaagggtcctttctct -0.4051230
14 CDC42BPG_101_6052 CDC42BPG gaggagctgccaccatcccg -0.4346633
11 CDC42BPG_101_12104 CDC42BPG gggtaccacctcccctccag -0.5207291
1 CDC42BPG_0_0 CDC42BPG ggccctaacagagggcatca -0.5829203
3 CDC42BPG_0_6052 CDC42BPG ggcccatcatcttgagctgt -0.6444921
12 CDC42BPG_101_15130 CDC42BPG gctgggttatgtccacaggt -0.6687819
9 CDC42BPG_100_6052 CDC42BPG ggagctgccaccatcccgcg -1.2168444

CHEK1

In addition to the plots below, the following information has been retrieved via biomaRt:

ENSEMBL ID (links to Ensembl) ENSG00000149554

HGNC SYMBOL (links to GeneCards) CHEK1

GENE DESCRIPTION
checkpoint kinase 1 [Source:HGNC Symbol;Acc:HGNC:1925]

GO TERM
replication fork

MIM GENE DESCRIPTION
CHECKPOINT, S. POMBE, HOMOLOG OF, 1; CHEK1;;CELL CYCLE CHECKPOINT KINASE; CHK1

ENSEMBL PROTEIN ID
ENSP00000388648

PROTEIN FAMILY DESCRIPTION
SERINE/THREONINE KINASE CHK1 EC_2.7.11.1 CHK1 CHECKPOINT HOMOLOG CHECKPOINT KINASE 1

Wilcox p-value: 0.9671744

DESeq2 p-value: 0.1231337

MAGeCK p-value: 0.701894

designs genes sequence log2Foldchange
8 CHEK1_116_0 CHEK1 gatgttgccgctcccgccca 0.8004400
15 CHEK1_132_43812 CHEK1 gaaatatgcctaggcatggt 0.4073559
4 CHEK1_101_0 CHEK1 gaccgcgctgcgcttccagg 0.3253318
1 CHEK1_0_14604 CHEK1 gagccacaatgccctgccat 0.2268288
10 CHEK1_11_0 CHEK1 ggagctggttcacagaaaaa 0.1493467
3 CHEK1_100_21906 CHEK1 gttgttgatatagtaaccta 0.0000000
2 CHEK1_0_29208 CHEK1 gtaagcaaaactttggggaa -0.0097152
12 CHEK1_120_0 CHEK1 gaatgtcggcggctccaaaa -0.0418202
11 CHEK1_11_7302 CHEK1 ggatatcaacatgacttttg -0.0444676
14 CHEK1_12_0 CHEK1 gagggcagaatttcaacctt -0.0732607
5 CHEK1_105_0 CHEK1 gcccaggcctccaagcctca -0.3542135
9 CHEK1_118_0 CHEK1 gcggctccaaaagggtgacg -0.4289122
6 CHEK1_10_0 CHEK1 gtaaagcattgttttggagc -0.5804622
13 CHEK1_121_0 CHEK1 gcttttcccgccaaaacttg -0.7653539
7 CHEK1_115_0 CHEK1 gcccagggctgctttgcagt -1.4143340
16 CHEK1_13_0 CHEK1 gcaagagctgttaattttcg -1.6893490

CSF1R

In addition to the plots below, the following information has been retrieved via biomaRt:

ENSEMBL ID (links to Ensembl) ENSG00000182578

HGNC SYMBOL (links to GeneCards) CSF1R

GENE DESCRIPTION
colony stimulating factor 1 receptor [Source:HGNC Symbol;Acc:HGNC:2433]

GO TERM
CSF1-CSF1R complex

MIM GENE DESCRIPTION
COLONY-STIMULATING FACTOR 1 RECEPTOR; CSF1R;;MCSFR;;ONCOGENE FMS; FMS;;c-FMS;;CD115 ANTIGEN; CD115;;V-FMS MCDONOUGH FELINE SARCOMA VIRAL ONCOGENE HOMOLOG, FORMERLY

ENSEMBL PROTEIN ID
ENSP00000445282

PROTEIN FAMILY DESCRIPTION
MAST/STEM CELL GROWTH FACTOR RECEPTOR KIT PRECURSOR SCFR EC_2.7.10.1 PROTO ONCOGENE C KIT TYROSINE KINASE KIT

Wilcox p-value: 0.9671744

DESeq2 p-value: 0.1115685

MAGeCK p-value: 0.701894

designs genes sequence log2Foldchange
15 CSF1R_117_25749 CSF1R ggcctcggtggggaagtggc 0.7432286
7 CSF1R_109_17166 CSF1R gtctgggcccattcagcaca 0.6694507
1 CSF1R_0_0 CSF1R gtgtggccagccacatgcca 0.1437269
9 CSF1R_10_0 CSF1R gataggacagaggatgccca 0.0229662
5 CSF1R_103_17166 CSF1R ggcctgtcctaaagagacct -0.0440560
12 CSF1R_114_25749 CSF1R gctcttaccatgccaagctg -0.0897533
4 CSF1R_101_17166 CSF1R gcttcaggcggggcagagag -0.1942767
6 CSF1R_106_17166 CSF1R gagggtgaaggtgtgcctgc -0.3364257
10 CSF1R_10_25749 CSF1R ggcagggtctagagtagagg -0.3598989
11 CSF1R_111_0 CSF1R gtgcagctcctgactattcc -0.4741526
8 CSF1R_109_8583 CSF1R gactcatgttggcatacagt -0.5012091
2 CSF1R_0_25749 CSF1R gggcactgcattgatagtcc -0.5221593
3 CSF1R_100_17166 CSF1R gccctgggatgactgagacc -0.6523193
13 CSF1R_115_25749 CSF1R gccaagctgtggccaccagc -0.8383244
14 CSF1R_116_8583 CSF1R gacatgcaggcgaccaccac -0.8513914

PRKACB

In addition to the plots below, the following information has been retrieved via biomaRt:

ENSEMBL ID (links to Ensembl) ENSG00000142875

HGNC SYMBOL (links to GeneCards) PRKACB

GENE DESCRIPTION
protein kinase, cAMP-dependent, catalytic, beta [Source:HGNC Symbol;Acc:HGNC:9381]

GO TERM
transferase activity, transferring phosphorus-containing groups

MIM GENE DESCRIPTION
PROTEIN KINASE, cAMP-DEPENDENT, CATALYTIC, BETA; PRKACB

ENSEMBL PROTEIN ID
ENSP00000378314

PROTEIN FAMILY DESCRIPTION
CAMP DEPENDENT KINASE CATALYTIC SUBUNIT PKA C EC_2.7.11.11

Wilcox p-value: 0.9671744

DESeq2 p-value: 1.054877e-06

MAGeCK p-value: 0.701894

designs genes sequence log2Foldchange
11 PRKACB_122_114595 PRKACB gataattttatatcccacat 0.4588957
1 PRKACB_0_114595 PRKACB gctacaataaggcagtggat 0.2836619
5 PRKACB_117_91676 PRKACB gtgaaaacatttcaccccca 0.0988086
12 PRKACB_125_22919 PRKACB gccaaatcatcttatatgtc 0.0802308
9 PRKACB_120_114595 PRKACB gttcatatcaaaagttctgc 0.0000000
13 PRKACB_127_137514 PRKACB gtctgcaagggccaattctt 0.0000000
4 PRKACB_117_114595 PRKACB ggttggtctgcaaagaatgg -0.0384741
10 PRKACB_121_114595 PRKACB gtcattcttctaaggtgccc -0.1305172
7 PRKACB_11_0 PRKACB gacatgttagcacttaaagt -0.3644069
8 PRKACB_11_137514 PRKACB gggagacatcctcactttcc -0.4000529
6 PRKACB_118_22919 PRKACB gcactactagcattgcatga -0.6499614
14 PRKACB_134_137514 PRKACB gtacacactgctgctagtga -0.7959443
3 PRKACB_105_0 PRKACB gccggtgctaaggagttcgc -0.9016024
15 PRKACB_137_68757 PRKACB gtcaggagagtacttctacc -0.9260958
2 PRKACB_101_91676 PRKACB gttggatcattacagacagc -0.9308058

NRG3

In addition to the plots below, the following information has been retrieved via biomaRt:

ENSEMBL ID (links to Ensembl) ENSG00000185737

HGNC SYMBOL (links to GeneCards) NRG3

GENE DESCRIPTION
neuregulin 3 [Source:HGNC Symbol;Acc:HGNC:7999]

GO TERM
embryo development

MIM GENE DESCRIPTION
NEUREGULIN 3; NRG3

ENSEMBL PROTEIN ID
ENSP00000441201

PROTEIN FAMILY DESCRIPTION
PRO NEUREGULIN 3 MEMBRANE BOUND PRECURSOR PRO NRG3 [CONTAINS NEUREGULIN 3 NRG 3 ]

Wilcox p-value: 0.9671744

DESeq2 p-value: 0.0723033

MAGeCK p-value: 0.701894

designs genes sequence log2Foldchange
11 NRG3_1458_953040 NRG3 gagggccttgtccaaaattc 0.7452361
5 NRG3_1334_953040 NRG3 gcctcatatcgatgcatcat 0.5369936
15 NRG3_1482_635360 NRG3 gctgttcaaactcccaaaga 0.1508644
14 NRG3_1478_0 NRG3 gctgccgatatgtaaatgaa 0.0479055
7 NRG3_1366_953040 NRG3 gtggcactttcagctgctct 0.0444479
4 NRG3_1193_476520 NRG3 ggccagttgtcttcaaaccc 0.0000000
10 NRG3_1445_635360 NRG3 gccataacagatgttcctct 0.0000000
2 NRG3_1160_794200 NRG3 gggctagctctctaaccata -0.0736764
6 NRG3_1364_317680 NRG3 gcgctcaaaccctttactgg -0.4276937
12 NRG3_1470_0 NRG3 gacctcagatggaaccagaa -0.5988079
9 NRG3_140_953040 NRG3 gaatcctaggtccctatacc -0.6191415
13 NRG3_1476_0 NRG3 gcagttctccagtccatgac -0.6572912
3 NRG3_1167_317680 NRG3 gaactcaacgtagaccatct -0.6823694
8 NRG3_1369_794200 NRG3 gtctgctacagtgtctcact -0.7381306
1 NRG3_1156_158840 NRG3 ggccagaatctctagcactt -0.8424093

MAPK7

In addition to the plots below, the following information has been retrieved via biomaRt:

ENSEMBL ID (links to Ensembl) ENSG00000166484

HGNC SYMBOL (links to GeneCards) MAPK7

GENE DESCRIPTION
mitogen-activated protein kinase 7 [Source:HGNC Symbol;Acc:HGNC:6880]

GO TERM
negative regulation of NFAT protein import into nucleus

MIM GENE DESCRIPTION
MITOGEN-ACTIVATED PROTEIN KINASE 7; MAPK7;;PROTEIN KINASE, MITOGEN-ACTIVATED, 7; PRKM7;;EXTRACELLULAR SIGNAL-REGULATED KINASE 5; ERK5

ENSEMBL PROTEIN ID
ENSP00000311005

PROTEIN FAMILY DESCRIPTION
MITOGEN ACTIVATED KINASE 7 MAP KINASE 7 MAPK 7 EC_2.7.11.24 BIG MAP KINASE 1 BMK 1 EXTRACELLULAR SIGNAL REGULATED KINASE 5 ERK 5

Wilcox p-value: 0.9671744

DESeq2 p-value: 0.1051081

MAGeCK p-value: 0.701894

designs genes sequence log2Foldchange
9 MAPK7_10_4986 MAPK7 ggactgtggggagaggttcc 1.3398243
6 MAPK7_10_1662 MAPK7 gatggccacctgctggcctg 0.8788394
11 MAPK7_11_1662 MAPK7 gtcaccacatcgaaagcatt 0.2509378
4 MAPK7_0_4986 MAPK7 gtgttctcaggcacaccaaa 0.1995151
14 MAPK7_11_4155 MAPK7 ggccaaaggaaaatgggttc 0.0717681
8 MAPK7_10_4155 MAPK7 ggtctgggccaaaggaaaat 0.0412438
2 MAPK7_0_3324 MAPK7 gcgcattaaggaggccattg -0.2006852
7 MAPK7_10_3324 MAPK7 gggagacttggacttggaca -0.3209398
12 MAPK7_11_2493 MAPK7 ggtgactttggtatggctcg -0.3341234
10 MAPK7_10_831 MAPK7 ggctcggttctctgaaactc -0.4125705
3 MAPK7_0_4155 MAPK7 gttggaacgctggactcgaa -0.5507305
5 MAPK7_0_831 MAPK7 gcctacccctcccccgaccc -0.6895103
15 MAPK7_11_4986 MAPK7 gtgcaggagacatgcacctg -0.9092088
13 MAPK7_11_3324 MAPK7 ggctcctgagcctcggaagc -0.9935284
1 MAPK7_0_1662 MAPK7 gccccgcaggggagttgtga -1.0384330

BCL2L12

In addition to the plots below, the following information has been retrieved via biomaRt:

ENSEMBL ID (links to Ensembl) ENSG00000126453

HGNC SYMBOL (links to GeneCards) BCL2L12

GENE DESCRIPTION
BCL2-like 12 (proline rich) [Source:HGNC Symbol;Acc:HGNC:13787]

GO TERM

MIM GENE DESCRIPTION
BCL2-LIKE 12; BCL2L12

ENSEMBL PROTEIN ID
ENSP00000478415

PROTEIN FAMILY DESCRIPTION
BCL 2 12 BCL2 L 12 BCL 2 RELATED PROLINE RICH

Wilcox p-value: 0.9671744

DESeq2 p-value: 0.01822949

MAGeCK p-value: 0.701894

designs genes sequence log2Foldchange
7 BCL2L12_36_3576 BCL2L12 gaccagcttgctgcgcaggg 0.9538659
5 BCL2L12_34_3576 BCL2L12 ggggtccgaggccagctgta 0.8589222
13 BCL2L12_8_4768 BCL2L12 ggcccgcctggccctagcca 0.2415422
9 BCL2L12_38_3576 BCL2L12 ggcggaccagcttgctgcgc 0.1920837
1 BCL2L12_15_7152 BCL2L12 gccacttttcccttccagga 0.0261568
12 BCL2L12_42_4768 BCL2L12 gctccacgctgagtccggcc -0.1073999
10 BCL2L12_38_7152 BCL2L12 gccctcctggaagggaaaag -0.1755512
2 BCL2L12_32_3576 BCL2L12 ggccagctgtagggtggaag -0.4318455
11 BCL2L12_39_7152 BCL2L12 gccaggatgccctcctggaa -0.5109398
3 BCL2L12_32_4768 BCL2L12 gctccatggctagggccagg -0.7531135
4 BCL2L12_33_3576 BCL2L12 gtccgaggccagctgtaggg -0.7994554
6 BCL2L12_35_3576 BCL2L12 gctgcgcagggcggggtccg -0.9113653
8 BCL2L12_37_3576 BCL2L12 gcggaccagcttgctgcgca -1.9067631

ITPKA

In addition to the plots below, the following information has been retrieved via biomaRt:

ENSEMBL ID (links to Ensembl) ENSG00000137825

HGNC SYMBOL (links to GeneCards) ITPKA

GENE DESCRIPTION
inositol-trisphosphate 3-kinase A [Source:HGNC Symbol;Acc:HGNC:6178]

GO TERM
positive regulation of dendritic spine morphogenesis

MIM GENE DESCRIPTION
INOSITOL 1,4,5-TRISPHOSPHATE 3-KINASE A; ITPKA

ENSEMBL PROTEIN ID
ENSP00000260386

PROTEIN FAMILY DESCRIPTION
INOSITOL TRISPHOSPHATE 3 KINASE EC_2.7.1.127 INOSITOL 1 4 5 TRISPHOSPHATE 3 KINASE IP3 3 KINASE IP3K INSP 3 KINASE

Wilcox p-value: 0.9671744

DESeq2 p-value: 0.1123445

MAGeCK p-value: 0.701894

designs genes sequence log2Foldchange
3 ITPKA_0_7250 ITPKA gctgatcctgaagcgctgct 0.7576987
9 ITPKA_12_5800 ITPKA gtgtacttcagagaggacac 0.5788964
14 ITPKA_15_5800 ITPKA gggatgctctcaagattagc 0.4267269
4 ITPKA_0_8700 ITPKA gcgaggacggctatttgctg 0.3086007
5 ITPKA_10_1450 ITPKA ggccaatttagggcttcccc 0.0961843
15 ITPKA_16_2900 ITPKA gtgtcttcaaggcttccgaa 0.0250445
13 ITPKA_15_2900 ITPKA ggcatgagcaggtgtcttca 0.0000000
12 ITPKA_14_8700 ITPKA ggaggctctgccctctccag -0.0364790
2 ITPKA_0_2900 ITPKA gaggatgttcaagcttctcc -0.0990264
7 ITPKA_11_2900 ITPKA ggcttttcctaaccagccag -0.2495652
6 ITPKA_10_8700 ITPKA ggaccaggtgccagccacta -0.5905395
11 ITPKA_13_8700 ITPKA gggcaaagggcttcttcctc -0.7691593
1 ITPKA_0_0 ITPKA gcgaggacgtgggtcaggta -0.8777683
10 ITPKA_12_8700 ITPKA gccaggggttttgcccaccc -0.9386408
8 ITPKA_11_8700 ITPKA gaccaggtgccagccactaa -0.9836681
16 ITPKA_16_7250 ITPKA ggacatgtacaagaaaatgc -1.0849872

SGK2

In addition to the plots below, the following information has been retrieved via biomaRt:

ENSEMBL ID (links to Ensembl) ENSG00000101049

HGNC SYMBOL (links to GeneCards) SGK2

GENE DESCRIPTION
serum/glucocorticoid regulated kinase 2 [Source:HGNC Symbol;Acc:HGNC:13900]

GO TERM
positive regulation of transporter activity

MIM GENE DESCRIPTION
SERUM/GLUCOCORTICOID-REGULATED KINASE 2; SGK2

ENSEMBL PROTEIN ID
ENSP00000392795

PROTEIN FAMILY DESCRIPTION
SERINE/THREONINE KINASE EC_2.7.11.1 SERUM/GLUCOCORTICOID REGULATED KINASE

Wilcox p-value: 0.9671744

DESeq2 p-value: 0.04938286

MAGeCK p-value: 0.79024

designs genes sequence log2Foldchange
7 SGK2_103_8362 SGK2 ggctccccatctgtataatg 0.6581153
12 SGK2_107_0 SGK2 gttttccactaacgtcatag 0.3187377
1 SGK2_0_25086 SGK2 gagtccaggactggcaggac 0.1458818
10 SGK2_106_25086 SGK2 ggtgaacaagacttgaggaa 0.0372740
13 SGK2_107_25086 SGK2 gggtgaacaagacttgagga -0.0304471
2 SGK2_100_4181 SGK2 ggaacaccatctcaccctag -0.1618814
3 SGK2_101_0 SGK2 gcctctcacctgaaaactcc -0.2146417
4 SGK2_101_4181 SGK2 gactggagacttctgttccc -0.5254519
8 SGK2_104_0 SGK2 gctaaaaccgctatagtcct -0.5363070
5 SGK2_101_8362 SGK2 ggctttcaggaaaaccagcc -0.5808980
11 SGK2_106_4181 SGK2 gggggctagctgatgtcacg -0.6085201
9 SGK2_105_4181 SGK2 ggggctagctgatgtcacgc -0.9623739
6 SGK2_102_0 SGK2 gaatcctcagcactgagcac -1.2491888

PKMYT1

In addition to the plots below, the following information has been retrieved via biomaRt:

ENSEMBL ID (links to Ensembl) ENSG00000127564

HGNC SYMBOL (links to GeneCards) PKMYT1

GENE DESCRIPTION
protein kinase, membrane associated tyrosine/threonine 1 [Source:HGNC Symbol;Acc:HGNC:29650]

GO TERM

MIM GENE DESCRIPTION
PROTEIN KINASE, MEMBRANE-ASSOCIATED TYROSINE/THREONINE, 1; PKMYT1;;CDC2-INHIBITORY KINASE, MEMBRANE-ASSOCIATED TYROSINE/THREONINE, 1;MYT1

ENSEMBL PROTEIN ID
ENSP00000461628

PROTEIN FAMILY DESCRIPTION
UNKNOWN

Wilcox p-value: 0.9671744

DESeq2 p-value: 0.8398297

MAGeCK p-value: 0.79024

designs genes sequence log2Foldchange
2 PKMYT1_0_10722 PKMYT1 ggggtgacctccgcagcttc 0.8814200
13 PKMYT1_105_5361 PKMYT1 ggctggctcaccctgccagc 0.7427912
14 PKMYT1_105_7148 PKMYT1 gtgggtaccacctcccttcc 0.1817254
11 PKMYT1_104_5361 PKMYT1 gccagctggctacagcccct 0.1005956
4 PKMYT1_0_8935 PKMYT1 ggtgccactcagaggtggcg -0.0288452
3 PKMYT1_0_7148 PKMYT1 gctcaaagcctgccagcctg -0.0485978
7 PKMYT1_102_5361 PKMYT1 ggatgacgacagcctagggt -0.0722303
12 PKMYT1_104_7148 PKMYT1 ggaggacggccggctctatg -0.2154798
6 PKMYT1_101_7148 PKMYT1 ggcccgcaagttggccgagg -0.3074704
15 PKMYT1_106_5361 PKMYT1 ggcaggtaagctcccgacag -0.3181115
1 PKMYT1_0_0 PKMYT1 gcccggatgcacagttctga -0.3338034
10 PKMYT1_103_7148 PKMYT1 gcgttccatgtcaccattcc -0.4395002
5 PKMYT1_100_7148 PKMYT1 gcccgcaagttggccgaggt -0.4400416
9 PKMYT1_103_5361 PKMYT1 gggatgacgacagcctaggg -0.7124507
8 PKMYT1_102_7148 PKMYT1 gtcaccattccggggcccca -0.8879269

FASTK

In addition to the plots below, the following information has been retrieved via biomaRt:

ENSEMBL ID (links to Ensembl) ENSG00000164896

HGNC SYMBOL (links to GeneCards) FASTK

GENE DESCRIPTION
Fas-activated serine/threonine kinase [Source:HGNC Symbol;Acc:HGNC:24676]

GO TERM
protein phosphorylation

MIM GENE DESCRIPTION
FAS-ACTIVATED SERINE/THREONINE KINASE; FASTK;;FAST

ENSEMBL PROTEIN ID
ENSP00000444498

PROTEIN FAMILY DESCRIPTION
FAS ACTIVATED SERINE/THREONINE KINASE FAST KINASE EC_2.7.11.8

Wilcox p-value: 0.9671744

DESeq2 p-value: 0.1427661

MAGeCK p-value: 0.79024

designs genes sequence log2Foldchange
13 FASTK_10_3636 FASTK gccaatcccgaagcagctat 1.0609902
5 FASTK_0_3030 FASTK gcactggggcctcctccaac 0.4466181
10 FASTK_10_1818 FASTK ggccaggagcaccatcacgt 0.3911856
4 FASTK_0_2424 FASTK gagacatgtaaagtatcatg 0.3431926
3 FASTK_0_1818 FASTK ggtgtgtctgcttcaggaaa 0.3339876
9 FASTK_10_1212 FASTK gcatgctgctgacaggaggt 0.2198671
15 FASTK_11_0 FASTK gctggtagcccatcaggcct -0.1872616
1 FASTK_0_0 FASTK gggcctggagcttctgcctc -0.2910421
7 FASTK_0_606 FASTK gtccttgtgactgaggagaa -0.4000973
6 FASTK_0_3636 FASTK gacggtgctgggttccatgc -0.4481787
2 FASTK_0_1212 FASTK gccaggatcctctcaaggca -0.5365657
14 FASTK_10_606 FASTK gaggggtgcctgtggggagg -0.5394610
11 FASTK_10_2424 FASTK gagcaggagcaagcgttcct -0.5862362
16 FASTK_11_1212 FASTK ggaagcaggggtggcacccc -0.6413484
8 FASTK_10_0 FASTK gcaggagctggtagcccatc -0.7574409
12 FASTK_10_3030 FASTK gcgcgcccaccactactcgg -1.4239192

AK1

In addition to the plots below, the following information has been retrieved via biomaRt:

ENSEMBL ID (links to Ensembl) ENSG00000106992

HGNC SYMBOL (links to GeneCards) AK1

GENE DESCRIPTION
adenylate kinase 1 [Source:HGNC Symbol;Acc:HGNC:361]

GO TERM

MIM GENE DESCRIPTION
ADENYLATE KINASE 1; AK1;;ADENYLATE KINASE, SOLUBLE

ENSEMBL PROTEIN ID
ENSP00000448741

PROTEIN FAMILY DESCRIPTION
UNKNOWN

Wilcox p-value: 0.9671744

DESeq2 p-value: 0.1097052

MAGeCK p-value: 0.79024

designs genes sequence log2Foldchange
10 AK1_2_0 AK1 ggaacatatgctcagagcga 1.0119537
9 AK1_21_0 AK1 gcttaaagggagcccagctt 0.6911687
4 AK1_14_0 AK1 gaacggagggttgaggggct 0.3342489
14 AK1_3_0 AK1 gaacatatgctcagagcgat 0.0280782
11 AK1_37_0 AK1 gtcaaaaaatgaaagcgcgc 0.0000000
2 AK1_10_0 AK1 gggtccacaagaggctctct -0.0714469
7 AK1_1_0 AK1 ggatttcattcgggcaggcg -0.0968484
13 AK1_39_0 AK1 gcaggaagggttctccccag -0.1320204
3 AK1_12_0 AK1 gaggggtggctggcaaaggg -0.3372427
15 AK1_40_0 AK1 gggttctccccaggggattc -0.3427298
16 AK1_41_0 AK1 ggttctccccaggggattct -0.3996150
5 AK1_15_0 AK1 gttgaggggctggggacttg -0.4933369
12 AK1_38_0 AK1 gcgcaggaagggttctcccc -0.5140515
6 AK1_17_0 AK1 gcacatgaatcaactccaac -0.6116374
8 AK1_20_0 AK1 ggcttaaagggagcccagct -0.6754054
1 AK1_0_0 AK1 gtgcagtccaggccccggca -0.6771400

Compare Analysis

On the following pages, a comparison between the hit analysis methods is generated.

Enriched

List

Moreover, the 5 % top enriched hits sorted according to MAGeCK are stored in TRAILscreen-COMPARE-HITS.xls and are listed below:

wilcox.log2fc wilcox.pval deseq.log2fc deseq.pval mageck.fdr mageck.rank
CASP8 2.8382733 0.0178241 2.8539899 0.0000000 0.001650 1
BAX 2.4706303 0.0178241 2.0761255 0.0000000 0.001650 2
FADD 2.8332735 0.0184341 2.7466780 0.0000000 0.001650 3
PASK 1.3650980 0.0178241 1.3056495 0.0000001 0.003713 4
TYRO3 1.0116807 0.9671744 0.6568340 1.0000000 0.659241 5
AMHR2 1.4654422 0.9671744 1.5964273 0.0000000 0.659241 6
FN3K 1.4685534 0.4030853 1.2257973 0.0000044 0.661245 7
PRKY 1.2424191 0.9671744 1.1629272 0.0000322 0.785149 8
CDK5RAP3 0.8994121 0.4202992 0.6707720 0.4964897 0.785149 9
ROR2 0.6903593 0.9671744 0.4127949 1.0000000 0.785149 10
BMPR1A 1.6970986 0.9671744 1.6606699 0.0000000 0.785149 11
DGKI 1.0784097 1.0000000 0.8176321 0.0921921 0.785149 12
IP6K3 1.3336231 0.8019777 0.9775242 0.0004042 0.785149 13
SGK1 1.0084174 0.9671744 1.0027240 0.0169285 0.785149 14
CSNK1A1 0.9112430 0.9802939 0.2043810 1.0000000 0.785149 15
RPS6KA5 1.0125970 0.9671744 2.0613922 0.0000000 0.792340 16
FGFR2 0.9059273 0.9802939 0.6981811 0.2598086 0.792340 17
FER 0.9117190 0.9671744 0.8130390 0.0356230 0.792340 18
GAK 0.5387169 0.9671744 0.1504273 1.0000000 0.792340 19
MYC 0.9824296 0.9671744 1.0493447 0.0000603 0.819189 20
NME7 1.0651572 0.9671744 0.7293105 1.0000000 0.819189 21
MAP3K13 0.8027186 0.9802939 0.7890949 0.1618026 0.903465 22
CDK17 1.4075806 0.9671744 1.9749507 0.0000000 0.903465 23
UCK1 0.9366588 0.9671744 0.8130611 0.0389347 0.903465 24
SPA17 0.9005421 0.4030853 0.6442567 0.7318362 0.903465 25
STK32A 0.7202280 0.9671744 0.5301267 1.0000000 0.903465 26
MVK 0.8969374 0.9671744 0.5232427 1.0000000 0.912769 27
BIRC5 1.0329737 1.0000000 0.4600186 1.0000000 0.912769 28
MAP2K7 1.4371620 1.0000000 1.1032489 0.0001706 0.912769 29
TNFRSF8 0.9060177 0.9820183 0.8932955 1.0000000 0.919967 30
RIPK2 1.0454989 0.9671744 0.9771641 0.0006398 0.987129 31
MAP2K3 0.6951014 1.0000000 0.5789501 1.0000000 0.987129 32
CDK9 1.0234281 0.9820183 0.6566444 1.0000000 0.987129 33
DUSP10 0.9243816 0.9671744 0.8461876 0.6000355 0.987129 34
LMTK3 1.4188888 1.0000000 1.0154113 0.0001587 0.987129 35
TK2 0.8383299 0.9671744 0.4435478 1.0000000 0.987129 36
NLK 0.8116484 0.9671744 0.7749996 1.0000000 0.987129 37
PRKD2 0.8994031 0.9671744 0.5585946 1.0000000 0.987129 38
PRPS1 1.2220187 0.9802939 1.0154924 0.0010360 0.987129 39
SGK223 0.7552550 1.0000000 0.5012950 1.0000000 0.987129 40
SRPK2 0.7442764 0.9671744 0.8655405 0.0257051 0.987129 41

Depleted

List

Moreover, the 5 % top depleted hits sorted according to __MAGeCK_ are stored in TRAILscreen-COMPARE-HITS.xls and are listed below:

wilcox.log2fc wilcox.pval deseq.log2fc deseq.pval mageck.fdr mageck.rank
TNFRSF13C NA NA -0.0773572 1.0000000 0.701894 1
DYRK2 NA NA -0.1075289 1.0000000 0.701894 2
CDC42BPG -0.1892980 0.8749233 -0.9735155 0.0633304 0.701894 3
CHEK1 -0.0937255 0.9671744 -0.8384986 0.1231337 0.701894 4
RAPGEF3 NA NA NA NA 0.701894 5
TNFRSF11A NA NA NA NA 0.701894 6
CSF1R -0.1386295 0.9671744 -0.7419327 0.1115685 0.701894 7
PTK6 -0.0624989 0.9671744 -0.4113096 1.0000000 0.701894 8
BMF NA NA -0.3582229 1.0000000 0.701894 9
TESK2 NA NA -0.4762269 1.0000000 0.701894 10
NUAK2 NA NA NA NA 0.701894 11
AURKA NA NA -0.1312312 1.0000000 0.701894 12
PRKACB -0.2086010 0.9671744 -1.3268439 0.0000011 0.701894 13
NRG3 -0.1267339 0.9671744 -0.7790845 0.0723033 0.701894 14
TNFRSF13B NA NA -0.1269107 1.0000000 0.701894 15
AZU1 -0.1144076 0.9671744 -0.6156798 1.0000000 0.701894 16
WIF1 -0.0661615 0.9671744 -0.8396864 0.0242731 0.701894 17
MAPK7 -0.0149634 0.9671744 -0.7443061 0.1051081 0.701894 18
HK3 NA NA -0.8896877 0.0161867 0.701894 19
BCL2L12 -0.0770045 0.9671744 -0.8493349 0.0182295 0.701894 20
TP53BP1 NA NA -0.0432170 1.0000000 0.701894 21
ITPKA -0.0990443 0.9671744 -0.9804692 0.1123445 0.701894 22
JAK3 NA NA NA NA 0.701894 23
CSNK1A1L NA NA -0.4230571 1.0000000 0.736436 24
PAK7 NA NA -0.2202468 1.0000000 0.736436 25
MAP3K6 NA NA NA NA 0.790240 26
CAMK1G NA NA -0.4575927 1.0000000 0.790240 27
PFKFB1 NA NA -0.2941408 1.0000000 0.790240 28
SGK2 -0.1958993 0.9671744 -0.7987094 0.0493829 0.790240 29
PRKAA1 NA NA -0.5851408 1.0000000 0.790240 30
PKMYT1 -0.0488289 0.9671744 -0.6395897 0.8398297 0.790240 31
TSSK3 NA NA -0.4741866 1.0000000 0.790240 32
NTRK3 NA NA -0.1207013 1.0000000 0.790240 33
FASTK -0.0655226 0.9671744 -0.8297426 0.1427661 0.790240 34
AK1 -0.0566054 0.9671744 -0.7584758 0.1097052 0.790240 35
CLK3 NA NA -0.4754391 1.0000000 0.808760 36
MAPKAPK5 NA NA NA NA 0.808760 37
PTPRG NA NA -0.2841110 1.0000000 0.808760 38
CDC42SE2 NA NA -0.1443949 1.0000000 0.808760 39
ACVRL1 NA NA -0.4256669 1.0000000 0.808760 40
PAPSS1 NA NA -0.4826401 1.0000000 0.808760 41

Final Gene Table

A final table with all information for each gene is stored in
./data/TRAILscreen_FINAL.xls.

Annotate Hit Candidates

Hit candidates are annotated with additional information from biomaRt.

The following information is retrieved: Database: ensembl Dataset: hsapiens_gene_ensembl Filters: ensembl_gene_id description name_1006

The annotated list of all hit candidates is stored in

_ ./data _

** TRAILscreen_ANNOTATION.xls **

Data Extraction, Mapping and Files

Dataset .fastq file name Description
Control #1 2180_CRISPR_KO3_Drug_DMSO1_extracted-designs.txt Control #1
Control #2 2277_CRISPR_KO3_Drug_DMSO2_extracted-designs.txt Control #2
Treatment #1 2176_CRISPR_KO3_Drug_TRAIL1_extracted-designs.txt TRAIL #1
Treatment #2 2275_CRISPR_KO3_Drug_TRAIL2_extracted-designs.txt TRAIL #2

The data is located in
./data

and the script files for data extraction and mapping are located in
./scripts.

All file-based output (e.g. tables) from MAGeCK is stored in:
/Users/janwinter/Documents/PhD/GitHub/caRpools.

Parameter Value
Reverse Complement Sequence FALSE
Pattern of Data Extraction ACC(.{20})GT{2,4}AGAGC
Maschine Identifier FASTq M01100
Create Bowtie2 Index? FALSE
Reference .fasta File pilot-screen-library
Bt2 Threads 4
Bt2 Sensitivity very-sensitive-local
sgRNA Oligo Match perfect